JP2005511755A - A versatile method and apparatus for high-throughput production and purification of multiple types of proteins - Google Patents

Abstract

  Any of a variety of expression systems are used to screen and subsequently produce multiple types of proteins or peptides of interest, or other genetically expressed material. The multiple types of proteins are extracted from multiple types of separate processed green juices, and each type of green juice contains one type of target protein. A multi-channel apparatus processes multiple types of green juice by processing one type of green juice per channel. This device is computer controlled, which automatically controls the various valves and pumps in each channel to extract each target protein and deliver it into a dedicated storage container.

Description

  The present invention relates to a flexible and high throughput method and apparatus for expressing, extracting and purifying a relatively large amount of a given recombinant protein. The present invention further relates to a method and apparatus for purifying a plurality of types of predetermined proteins in an apparatus operating simultaneously in parallel separately. The present invention further relates to a method and apparatus for tracking, planning and managing a production system for simultaneously producing a plurality of types of predetermined proteins. The invention further relates to the production and purification of multiple types of proteins for use in personal pharmaceuticals. The invention further relates to the production and purification of multiple types of proteins for use in microarrays. The invention further relates to the production and purification of multiple types of proteins for use in protein-related studies.

  As both physicists and researchers recognize the importance of protein's role in physiological and metabolic functions in organisms such as humans, protein research and utilization is here in both the scientific and medical communities. It has been valued for decades. Various aspects of proteins are currently being studied, examples of which include protein-protein interactions, glycosylation, identification of protein disease-related labels, and other features. Proteins are used in microarrays for research and clinical applications, and large amounts of protein are required for antibody production and characterization. For this reason, protein production has become extremely important for further development in this field.

  There are many protein production techniques, each with its own advantages and limitations. Methods for producing full-length or partial-length proteins include bacterial expression systems, yeast expression systems, fungal expression systems, insect expression systems, mammalian expression systems, and GENEWARE (registered trademark) developed by Large Scale Biology Corporation of Vacaville, California. Plant expression systems.

  In all protein expression systems that express heterologous proteins, the cDNA or DNA sequence of interest is first cloned in a suitable vector. When this vector is transcribed or derived into a host species, the host is transformed with the vector DNA. For example, bacterial expression systems use plasmid, phage or virus-derived vectors for the expression of heterologous proteins. A vector DNA (insert DNA) containing a target nucleic acid sequence is inserted into a bacterium through standard transformation techniques such as a transformation technique using a calcium phosphate method and an electroporation method. In addition to these techniques, a number of kits are currently available for inserting isolated and purified insert DNA into a selected vector system. For this reason, bacterial expression systems are most widely used for routine expression and purification of heterologous proteins. Thus, although bacterial expression systems are often used for the expression of relatively large amounts of heterologous proteins, the problem of lack of proper folding and post-translational processing remains, resulting in functionally inactive molecules. There is a possibility. Thus, conventional bacterial expression systems are only suitable for small amounts of a narrow range of proteins.

  In insect expression systems, and not as much as in insect expression systems, yeast expression systems may provide folding, post-translational modifications and oligomerization similar to those found in natural heterologous proteins, but at the complexity they are natural. Not far from protein. One example of an insect expression system for protein production is the use of baculovirus in insect cells. Expression systems using plasmid-based Drosophila cells can also be used. This expression system does not require baculovirus manipulation and management. In both the baculovirus expression system and the plasmid system Drosophila expression system, a vector similar to the bacterial expression system is used to insert a heterologous protein into a host cell for continuous expression. Also in the case of a yeast expression system, commercially available DNA vectors such as pESC, pYES, pNMT, pYD, and pPICopGAP are used.

  In mammalian expression systems such as mammalian cell cultures transfected with plasmids or phage-based vectors or infected with viral vectors (eg, NIH3T3, HeLa, K562, 293 and other cell cultures), substantial post-translational Modifications can be made. Examples of commercially available vectors for use in mammalian expression systems include adeno-associated viruses, pFB retroviral vectors and viruses such as adenovirus, pACT, pBIND, pCAT, pCI, phRG-CMV, phRG-TK, phRL-TK, pSI And plasmids such as pERV, and phage-based vectors such as pBK, pBK-CMV and pBK-RSV. However, in the case of mammalian cells, culture-intensive work is required for the expression of heterologous proteins, and thus more problems may arise in order to expand to mass production. In addition, skilled artisans may be required to produce sufficient cells containing the desired amount of protein. This is because, for example, in the case of mammalian cells in particular, the efficiency of transient transfection is poor, which may require stable transformation of vector DNA and chromosomal integration.

  Proteins expressed in plant expression systems also require vectors for heterologous protein expression. For example, US Pat. Nos. 5,316,931 and 5,589,367 granted to Donson et al. Illustrate plant viral vectors suitable for systematic expression of heterologous genetic material in plants. The entire contents of these patents are hereby incorporated by reference. Donson et al. Describe plant viral vectors with heterologous subgenomic promoters for systematic expression of heterologous genes. Since such a recombinant plant virus vector can be used, the target protein and peptides can be produced in a plant host by recombination.

  It is also well known to isolate proteins produced in bacteria, yeast, insects (baculovirus) and mammalian culture. For example, Qiagen, Valencia, Calif. Sells metal affinity resins and magnetic beads that can be used in a 96-well plate format used for purification of 6 × His tag proteins. Such purification techniques are described in “A Handbook For High Level Expression And Purification Of 6 × His-tagged Proteins” (Qiagen March 2001), as well as US Pat. Nos. 4,877,830 and 5,047. No. 513, No. 5,284,933, and No. 5,310,663. The entire contents thereof are cited in this specification. However, many of the methods disclosed in the above-mentioned patents and the materials sold by Qiagen are optimized for the isolation of protein amounts measured in units of micrograms (not in mg) ), Not specifically designed for the purification of proteins produced in plants.

  Several treatments for isolating proteins, peptides and viruses from plants have also been described in the literature so far (US Pat. No. 4,400,471 to Johal, granted to Johal). U.S. Pat. No. 4,334,024, U.S. Pat. No. 4,268,632 to Wildman et al., U.S. Pat. No. 4,289,147 to Wildman et al., U.S. Pat. U.S. Pat. No. 4,347,324, U.S. Pat. No. 3,637,396 granted to Hollo et al., U.S. Pat. No. 4,233,210 granted to Koch, U.S. Pat. No. 4, granted to Koch 250, 197, the entire disclosures of which are incorporated herein by reference).

  A method system for mass purification of bioactive species produced in plants with good cost performance has been developed so far. Such biologically active species include proteins or peptides, particularly recombinant proteins or peptides, or virus particles, particularly viruses that have undergone genetic recombination. Specifically, US Pat. No. 6,037,456 issued to Garger et al. Describes a method for isolating and purifying large amounts of protein extracted, for example, from tobacco plants infected with recombinant tobacco mosaic virus. It is disclosed. The method disclosed in US Pat. No. 6,037,456 is primarily for proteins obtained from large amounts of tobacco plants or other acceptable plant material, where the amount of protein isolated can be from a few hundred grams to kilograms. For isolation and purification purposes. Also, pending US Patent Application No. 09 / 970,150, filed Oct. 3, 2001, which has the same assignee, “Flexible Processing Appraising for Purifying Viruses, Soluble Proteins and Peptoids. Discloses an automatic apparatus for purifying a large amount of protein produced in a plant. However, in this case as well, the amount of protein isolated is in the unit of several hundred grams to kilograms. The methods described in the above patents and pending patent applications have a number of advantages, but because the methods are intended for mass production of materials, the amounts of various proteins are in units of micrograms to milligrams, It is not easy to apply to the isolation of a smaller amount of multiple proteins. U.S. Patent No. 6,037,456 and U.S. Patent Application No. 09 / 970,150 filed Oct. 3, 2001, the same as assignee, "Flexible Processing Apparatus for Purifying Viruses and Pursuing Viruses". , Soluble Proteins and Peptides from Plant Sources, the entire contents of both are incorporated herein by reference.

Problems to be solved by the invention and means for solving them

  Therefore, the aim was to produce and purify multiple types of proteins that can be produced in any of a variety of cultures and that can be purified in a reliable manner to provide the desired amount of each protein. There is a need for a highly flexible expression system. There is also a need for a method and apparatus for efficiently producing and isolating 100 μg to several mg of recombinant protein obtained from plant material having a starting biomass of less than 10 g to 10 kg. Further, there is a need for methods and devices that efficiently produce and isolate similar amounts of recombinant protein produced in bacterial, insect, mammalian and / or yeast cultures. There is also a need for a method and apparatus that can efficiently perform protein production and isolation of a protein associated with a protein of interest for determining the structure and relationship of the proteome in a defined cell, tissue or host organism.

  At present, human genome research has progressed, opening the way to a new paradigm for implementing medical care that will surely change medical forms. If individualized drugs, the use of label-assisted diagnostics, and targeted therapies derived from the individual's molecular profile can be implemented, these can affect the way pharmaceuticals are developed and practiced. If so, the traditional linear process for drug discovery and development could soon be replaced by an integrated heuristic approach. Pharmaceutical companies currently rely on statistical evidence to prove that the target drug product can treat only a portion of the patient population due to adverse reactions that are undesirable for other people in the target patient population. The company manufactures a large amount of the single preparation. There is a need for small scale drug manufacturing methods where the drug is manufactured to fit a particular individual or patient population.

  If the virus or protein to be isolated is intended to be manufactured as a pharmaceutical product, a consistent and verifiable methodology is needed. Therefore, there is a need for an automated methodology and apparatus for isolating proteins, where an automated apparatus monitors the methodology used in the isolation process and provides its tracking and verification.

  The present invention relates to a multichannel apparatus for simultaneously purifying a plurality of types of proteins in parallel.

  The present invention also relates to a method and apparatus for simultaneously producing and purifying multiple types of proteins.

Definitions In order to provide a clear and consistent understanding of the specification and claims of this application, including the scope of the following terms, the terms are defined as follows.

  GENEWARE (R) is a technology developed by Large Scale Biology Corporation of Vacaville, California to test the function of a new gene and the protein encoded by the gene and to produce the protein in large quantities. GENEWARE (R) includes the use of vectors modified from viruses to place any gene or multiple genes in the test organism. Let the organism produce the protein product of the gene and study, collect and purify the product.

  Preferably, GENEWARE (R) uses tobacco plants infected with the transgenic tobacco tobacco virus or tobacco species associated therewith. The rapid growth of tobacco plants provides a very useful model organism for studying plant genes, and the high profitability of producing large quantities of either animal or plant proteins. For reasons, an example of GENEWARE (R) technology usually includes the use of tobacco plants. Various aspects of GENEWARE® technology are described in US patents assigned to Large Scale Biology Corporation (US Pat. No. 5,316,931 assigned to Donson et al., US Pat. No. 5,589 assigned to Donson et al.). No. 367, No. 5,766,885 granted to Carrington et al. No. 5,811,653 granted to Turpen et al. No. 5,866,785 granted to Donson et al., Donson No. 5,889,190 granted to Turpen et al. No. 5,889,191 granted to Turpen et al. No. 5,922,602 granted to Kumagai et al. No. 5,965,794 and No. 6,054,5 granted to Donson et al. Disclosed in No. 6). The entire contents thereof are cited in the present specification. However, it should be understood that the GENEWARE (registered trademark) technology can also be applied when using plants other than tobacco, such as corn and rice.

  In the following description, the terms “biomass”, “biomaterial” and “plant source” all extract or isolate the harvested plant, seed or target material such as viruses, proteins and / or peptides thereof. All plant parts that can be processed to do. For example, biomaterial treatment can include many types of plants and plant parts such as seeds, flowers, stalks, stems, roots, tubers, and leaf parts of plant material. Normally, fleshy and multi-leafed tobacco plants are ideal for mass production of certain proteins using GENEWARE (registered trademark) technology. From the following explanation, plants other than tobacco also use GENEWARE (registered trademark) technology It should be understood that it can be used for the production of proteins.

  Alternatively, corn, rice, cereal plants or other desirable plants can be used to produce the proteins and peptides of interest.

  In the following description, the terms “biomass” and “biomaterial” also refer to proteins produced in bacterial expression systems, insect expression systems, mammalian expression systems and yeast expression systems, and produced according to the methodologies described below. May refer to biological material collected for purification.

  The term “green juice” refers to a liquid extracted from a treated biomaterial. However, it should be understood that this green juice may refer to any liquid extracted from plant material or biomaterial, regardless of the color of the extracted liquid. For example, if one or more proteins are produced using the Large Scale Biology Corporation's GENEWARE (registered trademark) technology, green juice can be literally green because it is obtained from biomaterials such as collected tobacco There is. However, when the target protein is expressed in a bacterial expression system, an insect expression system, a mammalian expression system, a fungal expression system, or a yeast expression system, the liquid extracted from it can be other than green. It may be.

  As used herein, “virus” includes a virion comprising an infectious nucleic acid sequence combining one or more types of viral structural proteins and a non-infectious nucleic acid combining one or more types of viral structural proteins. Non-infectious virions and aggregates of viral structural proteins that do not contain nucleic acid sequences, that are not combined with nucleic acid sequences and may contain virus-like hollow particles (VLPs) It is defined as including the group consisting of These viruses may be natural or derived from recombinant nucleic acid technology and include any virus-derived nucleic acid that can be adapted by design or selection for replication in whole plants, plant tissues or plant cells. .

  The “virus population” as used in this specification is defined as one or more types of viruses as defined above. This virus population consists of a heterologous selection that includes all combinations and proportions of the viruses.

As used herein, “virus-like hollow particles (VLPs)” are defined as self-assembled structural proteins. This structural protein is encoded by one or more nucleic acid sequences, which nucleic acid sequence (s) are inserted into the genome of the host viral vector.
“Proteins and peptides” are defined as either naturally occurring proteins and peptides, or recombinant proteins and peptides produced by transfection or transgenic transformation.

  The terms “target protein”, “target material” and “multiple types of target material” refer to any material, compound, organic structure or combination of materials to be isolated using the purification method and / or apparatus according to the present invention. Examples of target proteins or one or more target materials include, but are not limited to, virions, virus-like hollow particles, viruses, proteins and / or peptides, receptors, receptor antagonists, antibodies Single chain antibodies, enzymes, neuropolypeptides, insulin, antigens, vaccines, peptide hormones, calcitonin, and human growth hormone. In addition, the target protein or one or a plurality of types of target materials are combined with an antibacterial peptide or protein consisting of protegrin, magainin, cecropin, melittin, indolicidin, defensin, β-defensin, cryptdin, clavinin, plant defensin, herring and bactenine. You can also

  As used herein, "bacteria" is a fine single cell that proliferates by cell division, and that the cells are normally contained within the cell wall, occur in a spherical, rod-like, spiral or curved shape and can be found in virtually any environment Defined as including a group of microorganisms.

  As used herein, “bacterial culture” is defined as the maintenance and propagation of bacterial populations in vitro. This bacterial population is usually derived from a clone, ie from a single bacterial cell. Therefore, since all bacteria in a given bacterial culture contain the complement of the same gene, if it is a protein expression system, it should express the same heterologous protein sequence. However, in certain circumstances, this bacterial culture may contain multiple types of bacterial cells originating from one or more types of bacterial cells and containing different gene complements.

  “Mammalian cells” as used herein are defined as including a group consisting of cells derived from mammals. The starting mammalian cell includes, but is not limited to, tissue, fluid, blood, organ or biological source from a human or mammal.

  As used herein, “mammalian cell culture” is defined as including a group of cells derived from a mammal that can survive ex-vivo in a cell culture medium. This mammalian cell can be a primary cell derived directly from a mammalian cell. More typically, mammalian cells in mammalian cell culture can be immortalized, i.e., can be propagated and divided through an indefinite number of passages and divisions.

  As used herein, a group of small unicellular organisms that can grow and proliferate through budding, direct division (fission), or simply growing as an irregular flower (mycelium). Define as including. Yeast cells are those that can be transformed or transfected with a heterologous vector to express a nucleic acid sequence inserted within the heterologous vector. An example of a yeast cell is Saccharomyces cerevisiae, commonly used for transfection and expression of heterologous proteins.

  As used herein, “insect cells” are defined as including a group of cells derived from insects that can survive ex-vivo from an insect host. The insect cell can be transformed, transfected, or infected with the heterologous vector to express the protein sequence inserted within the heterologous vector. Examples of insect cells include High Five ™ cells, Sesjavka cells, Drosophila melanogaster cells, and Drosophila cells.

  An “affinity tag” is a molecule, ligand or polypeptide attached to a protein of interest (polypeptide). Examples of affinity tags include, but are not limited to, hexa-histidine, other metal tags, streptavidin, biotin, specific epitope tags for antibody purification, glutathione-S-transferase, □ -galactosidase, □ -amylase, and Other proteins or small molecule tags that can assist in the isolation and purification of the expressed protein.

  “Affinity matrix” refers to a solid material bound to a substrate or ligand. This material selectively binds to the affinity tag attached to the protein of interest. When the affinity tag and the affinity matrix are bound, the protein of interest remains in the column or other purification device. For this reason, the target protein can be separated therefrom if impurities are contained in the green juice. After washing the affinity matrix, the protein of interest attached with the affinity tag can be eluted from the column or other device in a substantially purified form. Examples of affinity matrices include agarose, cellulose, sepharose, sephadex and other chromatographic media, polystyrene beads, magnetic beads, filters, diaphragms, and other solid materials bound to substrates or ligands that bind to selected affinity tags. Can be mentioned.

A “histidine-labeled protein” is a protein of interest having a histidine affinity tag attached to either the carboxy terminus, the amino terminus, or the interior of the protein of interest. Usually, a histidine tag consists of six histidine moieties, but any combination or quantity thereof may be used. The histidine-labeled protein can be purified by binding the histidine-labeled protein to a metal affinity matrix such as Ni-NTAAAgarose (manufactured by QIAGEN, Inc.) and washing impurities from the bound affinity matrix. After purification, the histidine-labeled protein can be eluted from the column by competitive elution with imidazole using acid pH buffer conditions or by stripping the metal from the affinity matrix with EDTA (ethylenediaminetetraacetate).
Overview (Figure 1)
In accordance with the present invention, one or more proteins of interest are produced by any of a variety of methods, as shown in FIG.
1. According to one aspect of the present invention, a specific amount of one or more target proteins is automatically produced and purified in order to minimize the required materials and time and maximize the production of the target protein. Is done.

  Hereinafter, FIG. 1 will be briefly described. Details of the steps shown in FIG. 1 will be described later as appropriate.

  In the first step shown in S1 of FIG. 1, one or more types of target proteins are selected, and suitable vectors and inserts corresponding thereto are identified. This protein, vector, and insert selection process is further described in the section relating to FIG. 2 below.

  Hereinafter, the production and purification of at least one protein will be described in detail. In order to simplify the explanation, omit redundant parts, and make the explanation easier to understand, the contents included in most of the following are explanations on the production and purification of only one kind of protein. Should be understood from the following description that can be produced and purified simultaneously.

  After selecting the protein, vector, and insert, in S2 of FIG. 1, an organism or expression system suitable for the production test of the target protein is selected. Specifically, for example, any one or a plurality of expression systems composed of a bacterial expression system, an insect expression system, a mammalian expression system, a plant expression system, a fungal expression system, and a yeast expression system can be used.

  In S3 of FIG. 1, the protein produced as a result of the test in S2 of FIG. 1 is screened to determine whether the target protein was appropriately expressed by the organism or expression system used in the production test. In addition, the relationship between the amount of biomaterial produced and the amount of expressed protein is also determined. Although the amount of protein expressed at this stage is relatively small, it is screened using various functional and structural tests. Therefore, the screening process shown in S3 includes a plurality of sub-steps, which will be described in detail below.

  In S4 of FIG. 1, it is determined which expression system (bacteria, insect, mammal, plant, fungus or yeast) is optimal for the expression of the target protein. For example, the GENEWARE® technology is typically first used in the S2 test. If the protein of interest is not properly expressed, another organism such as a bacterial expression system is tested and screened as indicated at S3. When an expression system suitable for production of the target protein is constructed, the amount of biomaterial necessary to produce a desired amount of purified protein is calculated. This will be described in detail below. Alternatively, various protein expression systems can be tested in parallel, i.e., bacterial expression systems, plant expression systems, and insect expression systems can be tested at the same time to produce an appropriate expression system for greater expression and purification. You may decide.

  As shown in S5 of FIG. 1, the target protein is expressed in the determined optimal expression system or organism. The biomass produced by this optimal expression system is collected as shown in S6 of FIG. 1 and treated or pretreated before purification as shown in S7. This protein expression, collection and pretreatment steps will be described in more detail in the section relating to FIG. 3 below. The purification step shown in S7 will be described in more detail in the section relating to FIG. 4 below.

As shown in S8 of FIG. 1, the purified target protein is tested to confirm its characteristics and consistency.
Protein and insert selection (Figure 2)
There are many processes in which one or more types of target proteins can be selected for production and purification, depending on their function or purpose. For example, the protein or proteins of interest can be transferred to a patient, such as a vaccine, as described in pending US patent application Ser. No. 09 / 522,900, filed Mar. 10, 2000. It can be a unique drug. In this case, a sequence for expressing a specific protein is obtained from the patient's own DNA. Alternatively, the target protein can be a target protein for use in a microarray or so-called protein chip, in which case physiology from the collected sample (blood, formation, urine, sputum, cerebrospinal fluid or biological sample) Protein targets can be selected to enable assessment of genetic parameters, organ function or dysfunction, and identification of various pathological infection states.

  If a sequence is required to express a specific protein or a well-known protein (eg, a sequence that is not specifically taken from a patient), the sequence can be expressed using a computer system such as the example schematically shown in FIG. And can be retrieved from a variety of databases, including shared private ownership. In the case of FIG. 2, each of the databases can be searched by in-house customers A and B to N accessing the respective databases. Examples of publicly available databases include: National Center for Biotechnology Information (GenBank and BLAST) nucleotide and protein databases, European Molecular Biological Laboratory (SWISS-PROT) nucleotide and protein databases, and other nucleotide and protein databases Literature is mentioned. Examples of private databases include Human Protein Index (HPI), MEDE (Molecular Effects Of Drugs), MAP (Molecular Analysis and Pathology), and databases from many laboratories. These sources include information detailing the protein or organism constituents, and information comparing protein expression in diseased and non-diseased subjects, or in normal and abnormal subjects. May be included.

  Collection of genetic information, that is, isolation of the target nucleic acid can be performed by various methods including polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), colony screening, and nucleic acid synthesis. The databases and literature mentioned above not only allow researchers and clinicians to select proteins to be expressed for a given state, but also include nucleotide and protein sequence information, so they are suitable target probes Can be designed to isolate the target cDNA and the protein of interest.

  Consideration of probe design and reaction conditions for the purpose of isolation is important for isolating a specific target protein, regardless of whether the target protein is a known or unknown species. Probes for isolating the cDNA of interest can be designed based on protein or DNA sequence information obtained from the databases and literature described above. Alternatively, probes may be designed using tryptic peptide information obtained from previously unknown proteins that have been isolated by methods of fractionating and isolating 2D gels or other proteins. This probe can be synthesized utilizing standard phosphoramidite chemistry or nucleic acid synthesis chemistry while incorporating standard dideoxynucleotide compounds (dATP, dGTP, dCTP, dGTP). Alternatively, modified nucleotides that can hybridize to two or more types of dideoxynucleotides (dITP or other modified nucleotides) may be used. When a protein sequence is used as a template for a nucleic acid probe, the probe set can be composed of at least one pair of primers that encode one permutation of the nucleic acid sequence. Alternatively, since the amino acid code is degenerate, one or more pairs of primers encoding different permutations of the corresponding nucleic acid sequence may be used. For example, lysine is encoded by two codon sequences, AAA and AAG. Thus, a sequence incorporating the amino acid lysine will contain both changes in the probe at the lysine position. In addition to professional synthesis, nucleic acid fragments excised from larger nucleic acid sequences (eg, cloning vector fragments and other nucleic acid fragments) can be used as probes for target nucleic acid isolation.

  Probes can be similarly designed, although not the same as well-known protein sequences. Such probes can isolate related proteins. Related proteins are proteins that may be difficult to isolate using standard probe design techniques because their amino acid sequence composition may vary between individuals. Alternatively, DNA can be screened with nucleic acid probes using low stringency conditions. This allows isolation and purification of related but not identical DNA sequences. This nucleic acid probe can be used for RT-PCR isolation and cloning from mRNA and total RNA samples. This nucleic acid probe may also be used for genomic DNA cloning from total genomic DNA using PCR amplification or isolation schemes. The total RNA or genomic DNA may be isolated from animals, plants, or bacterial / microbial cells or tissues using standard RNA or DNA purification techniques. Examples of techniques include alkaline lysis, guanidinium isothiocyanate, CsCl density gradient, phenol / SDS, phenol / chloroform, glass-based or silica-based chromatography, or other methods including kits commercially available from various manufacturers. It is done. Total RNA can be further fractionated on an oligo dT column or resin to obtain mRNA containing poly A RNA. In addition, mRNA can be isolated directly from cell culture and tissue lysates using standard lysis protocols in combination with oligo dT column chromatography (alkaline lysis, detergent lysis, mechanical division and other lysis methods). Is also possible.

  The cDNA strand obtained by reverse transcription of RNA can be replicated using DNA polymerase or other available polymerase to obtain double stranded DNA. A variety of standard molecular biology techniques using DNA polymerase can amplify the double stranded DNA and insert and ligate the amplified cDNA into a suitable expression vector for further analysis. Alternatively, genomic DNA can be amplified directly by PCR using DNA polymerase or other available polymerases. As with the amplified cDNA, the amplified genomic DNA can be inserted and ligated into a suitable expression vector for further analysis.

  Another protocol for isolating the DNA sequence of interest is the synthesis of the insert sequence and its complementary binding strand through a standard DNA synthesis protocol. For example, complementary DNA strands can be synthesized using standard phosphoramidite chemistry. For cloning purposes, asymmetric restriction enzyme sequences may be incorporated into the synthetic strand and cloned directly into replication and / or expression vectors. Alternatively, blunt-end ligation of restriction enzyme linkers can be performed using a standard molecular biological ligation protocol after annealing the DNA strand. The DNA synthesis methodology can be used to synthesize and purify picograms, nanograms, micrograms, or milligram quantities that vary mostly with the length of the sequence. This avoids amplification artifacts that can be introduced with the DNA polymerase enzyme. DNA synthesis can also be combined with PCR amplification to amplify sufficient quantities for DNA insertion into a suitable vector and subsequent DNA replication.

Another method is to use colony screening of bacterial hosts containing multiple types of vector insertions. Vector insertions typically include multiple types of nucleic acid sequences isolated from specific host tissues, organs or conditions. For example, commercially available bacterial “libraries” are available that correspond to multiple types of vector inserts obtained from murine liver or mice that are phenotypic for a particular disease. The isolated probe as described above can be used to screen large numbers of bacterial clones transferred onto solid media such as nitrocellulose or nylon filters or membranes. When the bacterial clones on the solid medium are lysed, the DNA contained in each clone is denatured and binds to the medium. This replaces the colony pattern with the same pattern of bound DNA. This medium is then hybridized to a labeled probe that identifies a clone containing the DNA sequence of interest. As a result of this clone being isolated and amplified by mass cultivation, the DNA is isolated and excised for manipulation into other target vectors.
Selection of vector According to the present invention, as described above and as described in the above-mentioned patent assigned to the same assignee as the present application, the isolated target DNA sequence is inserted into the vector, Can be produced by any of a variety of methods including bacterial expression systems, insect expression systems, mammalian expression systems and yeast expression systems, or by using features of the GENEWARE® technology. it can. Specifically, in the GENEWARE® expression system, the virus is genetically engineered to contain the gene sequence or insert of interest specifically selected for the protein encoded by the vector, tag and virus. Is manipulated. The virus is then applied to broad-leaved plant tissues such as leaves of tobacco plants and infects the organism. The plant and virus cooperate to express a specific protein, which is extracted from the plant tissue and purified. Such a basic workflow of the method according to the present invention will be described in detail below together with a detailed description of the apparatus used to validate the method according to the present invention. In addition, a computer system is described that tracks this workflow in a manner that will be more clearly described below, and that assists in distinguishing various aspects of the process.

  According to the present invention, when using GENEWARE® technology, specific vectors and inserts are selected for insertion into tobacco mosaic virus or other suitable virus. As shown in S1 of FIG. 1, one type of insert is selected for a specific protein encoded by the gene sequence of the insert. As can be seen more clearly from the description below, multiple types of viruses can be used as if each virus contains one insert so that multiple types of proteins can be expressed simultaneously. In addition, one or more vectors are selected from various vectors for each insert for insertion into a virus. For example, since not all vectors function with each insert, multiple types of vectors may be tested and tested for expression of the desired protein.

  As described above, various cloning and expression vectors may be used depending on the host expression system used for use in protein expression and purification. Usually, cloning and expression vectors can only transfect, transform, or infect one specific host expression system (eg, plant or bacteria only). However, depending on the nature of the nucleic acid sequences contained therein, there are cloning and expression vectors that can transfect, transform, or infect multiple types of host expression systems. One skilled in the art will appreciate that vectors can be designed to transfect, transform, or infect various host expression systems. All vectors capable of expressing the nucleic acid sequence of the vector insert in the host after transfecting, transforming, or infecting the host expression system are considered within the scope of the present invention. It is done.

  As described above, the vector to be used is selected according to the host expression system planned in the purification process. For example, in the case of a plant expression system, a viral vector derived from an RNA virus or a DNA virus can be used to introduce and express a heterologous protein sequence. RNA viral vectors are preferred because of their high expression levels and wide host range. US Pat. No. 5,316,931 describes a plant viral vector having a heterologous subgenomic promoter that can systemically infect a plant host and stably transcribe or express foreign gene sequences in the plant host. Has been. The entire content of this patent is hereby incorporated by reference. Similarly, US Pat. No. 5,811,653 describes an RNA virus vector obtained from a group of tobacco viruses that can overexpress genes in tobacco plants. The entire content of this patent is hereby incorporated by reference. US Pat. No. 5,977,438 describes an RNA virus vector in which a foreign gene is fused to an RNA virus protein (eg, a coat protein) to produce a relatively large amount of the foreign protein as a fusion protein. The entire content of this patent is hereby incorporated by reference.

  One suitable embodiment may be an RNA virus vector obtained from the tobacco virus group. An example of this is found in the GENEWARE vector derived from tobacco mosaic virus. In this GENEWARE vector, the TMV replicase coding sequence is located upstream of the coding sequence for the TMV transition protein. The ORF (reading frame) of the cDNA that is the ligated 3 'end of the TMV translocation protein is transferred to the polypeptide coding sequence of hexa-histidine affinity tag or other affinity tag coding sequence at its 3' end or 5 'end. Combined in-frame. This addition of the affinity tag coding sequence into the cloning and expression vectors allows the protein to be removed from the complex mixture by binding the desired affinity tag protein to the affinity matrix and then washing it to remove any impurities. Can be purified. After purification, the protein and affinity tag can be eluted from the affinity matrix in a substantially pure form. When this vector is optimized to be expressed at higher levels in protoplasts and inoculated leaves, the vector is cloned into the polylinker region 5 'of the cDNA insertion site using multiple restriction enzyme sites and expressed. A stop sequence can be included to properly stop the processed protein. For example, a tobacco mosaic virus-derived vector can include a TMV replicase coding sequence. This sequence can substantially increase expression in protoplasts and inoculated leaves. In addition, restriction enzyme sites including EcoRI, BamHI, SmaI, SacI, NotI, XbaI, SpeI, XhoI, SapI and others can be included in the multiple cloning site polylinker sequence that flanks the insertion site of the desired nucleic acid sequence. . Not only tobacco virus vectors but also other RNA virus vectors can be used, such as, but not limited to, rice dwarf virus, wound tumor virus, turnip yellow mosaic virus (timovirus) Genus), rice wilt virus, cucumber mosaic virus (genus cucumber virus), barley yellow dwarf virus (genus luterovirus), tobacco ring-point virus (genus nepovirus), potato X virus (genus potexvirus), potato Y Virus (genus Potyvirus), tobacco necrotic virus, tobacco stem necrotic virus (genus Tobravirus), tomato bushy stunt virus (genus Tombus virus), pumpkin mosaic virus, brom mosaic virus (bromovirus) and other RNAs Viruses It is. RNA within single-stranded RNA viruses can be either positive (+) or negative (-) strand.

  The DNA viral vector can be used for subsequent inoculation and protein expression in the host plant. Examples of DNA viral vectors include, but are not limited to, caulimoviruses such as cauliflower mosaic virus, cassava latent virus, bean golden mosaic virus, Chloris striate mosaic virus, maize streak Viruses and other DNA viruses are mentioned. Alternatively, a vector with a plasmid of root cancer can be used for Ti-mediated plant transformation.

  As mentioned above, the vector can be used to support the cloning and purification of the protein of interest by affinity tag sequences (hexa-histidine, other metal affinity tags, streptavidin, specific epitope tags for antibody purification, glutathione-S- Transferase, □ -galactosidase, and other tags that can assist in the isolation and purification of the expressed protein) and multiple cloning site linker sequences. DNA or RNA viral vectors can also include a nucleic acid sequence encoding a signal peptide to direct expression of the foreign protein into the interstitial fluid or medium thereof. This limits the amount of endogenous protein secreted into the interstitial fluid portion by the plant host, thus simplifying purification and increasing its efficiency. As an example of this, it incorporates a sequence for encoding a rice α-amylase signal peptide that directs secretion of the chimeric protein into the interstitial space of infected leaves and other transfected plant parts. Or ligation is mentioned.

  In addition to plant viral vectors for plant transformation and subsequent expression and purification, mammalian and prokaryotic expression vectors are used for subsequent transfection and transformation into mammalian and prokaryotic hosts. be able to. One suitable embodiment can be a dual mammalian / E. Coli expression vector capable of transcription and subsequent expression in either a bacterial or mammalian host. An example of this is the expression vector MEV (mammalian expression vector). It contains a conventional restriction enzyme cloning site (BamHI, EcoRI, SmaI, NotI, etc.) as well as a polylinker site with a SapI / EarI cloning site. The mammalian CMV immediate early enhancer promoter unit is separated by an intron upstream of the bacterial promoter unit. A Shine-Dalgarno / Kodak sequence is included to increase the efficiency of expression. A histidine-tag coding sequence is also included to increase the isolation and purification efficiency of the expressed protein. It is expressed in E. coli only by the presence of the SupE / F site.

  Vectors can be constructed so that nucleic acid inserts can be simultaneously inserted into multiple vectors for testing in various expression systems. For example, vectors that can be expressed in mammalian expression systems, bacterial expression systems, and plant expression systems can include the same restriction enzyme sites in the linker region of the vector DNA. Thus, the cDNA insert will be cloned into the corresponding restriction enzyme sites of multiple vectors, including MEV and GENEWARE vectors. At the same time, the same frame arrangement of all vectors for a given cDNA insert is ensured.

  Similar to plant virus vectors, affinity tag sequences (hexa-histidine, other metal affinity tags, streptavidin, protein A, calmodulin binding protein (CBP), chitin binding domain (for the purpose of insertion and purification into other vector DNA) It may be desirable to incorporate CBD), specific epitope tags for antibody purification, and other tags that can assist in the isolation and purification of the expressed protein) and multiple cloning site linker sequences. A signal peptidic sequence that directs secretion of the expressed protein for packaging and subsequent secretion into the extracellular fluid matrix may be used to simplify the purification of the expressed protein and increase its efficiency. In addition, other gene sequences that enhance the packaging function of the vector can be incorporated into the vector sequence. One example of this incorporates granulocyte / macrophage-colony stimulating factor (GM-CSF) encoding gene sequences into mammalian expression vectors for proteins that can be used to generate antibodies and other immune responses (eg, vaccines). That is. GM-CSF recruits antigen-presenting cells (APC; dendritic cells and macrophages) and at the same time promotes the generation of stem cell growth factor. This may stimulate the immunomodulator system and allow the mammalian host to produce more antibodies with higher specificity and affinity.

  A protein complex bound to a target protein can also be isolated using an affinity tag. For example, a tandem affinity purification (TAP) tag system previously demonstrated in yeast (Rigaut et al., “1999 Nature Biotechnology 17”, pages 1030-1032; Gavin et al., “2002 Nature 415”, pages 141-147) Is used to isolate the proteome, the protein of interest contains its TAP tag. In the TAP system, a protein of interest is attached to two types of affinity tags (eg, protein A and CBP) separated by a specific TEV protease cleavage sequence. In order to express a target protein at a natural level in this yeast expression system, a DNA cassette encoding a TAP tag is integrated by homologous recombination into the genome of a single phase yeast cell in frame with the target protein. .

  The TAP system consists of a two-step purification system to reduce nonspecific binding. This affinity purification system can be combined with the presence of a specific TEV protease cleavage sequence to create mild elution conditions and increase the likelihood of isolating proteomes and protein complexes. Usually, TAP purification comprises a step of attaching a TAP gene cassette containing two types of affinity labeling coding sequences separated by a specific TEV protease cleavage sequence to the end of the target gene sequence in frame. The TAP gene cassette can be attached to the end of the protein coding sequence by PCR cloning or amplification, or insertion and ligation into a suitable vector containing the protein of interest. Next, the target TAP-tagged protein coding sequence is inserted into a host cell and expressed, and a protein related to the target protein is isolated and identified. Alternatively, the TAP gene cassette can be attached to the target protein in vitro by in-frame homologous recombination within the chromosome of the host organism. The TAP-tagged protein coding sequence is then expressed in vitro and the related protein is isolated.

  Isolation of related proteins is performed by a two-step purification process. First, a first affinity purification is performed to isolate all proteins associated with the TAP-labeled protein. Gentle elution is performed from the affinity matrix by cleavage with TEV protease, and the proteome or protein complex is eluted from the first affinity purification matrix. In order to remove all non-specific proteins, contaminants and TEV protease, a second affinity purification matrix is used to perform a second affinity purification. Next, the relevant protein is released from the bound target protein by EGTA elution. The isolated protein is further isolated using denaturing gel electrophoresis. Each protein band is digested with trypsin and analyzed with a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS). This protein is identified by well-known database search algorithms such as Profound ™ and Protein Prospector ™ in databases such as NCBI SWISS-PROT or other databases well known to those skilled in the art, and contains proteins within the proteome The amount as well as the protein content between the various proteome structures isolated can be analyzed.

Other mammalian, prokaryotic, insect, fungal or yeast vectors can also be used in combination with the methods and compositions disclosed herein. Examples include, but are not limited to, pBluescript, pCDNA3.1, pHAT, pIRES, pGBKT7, pVPack, pCMV-tag, pDual-GC, pBk-CMV, pIB-E, pMelBac, pureBac4, 5 / V5 -His, pYDl, pPIC9K, pYES2, pIB / V5-His, pIZT / V5-His, pIZ / V5-His, pNMTl, pPICZ, pNMTsl, pMET, pPIC3JK, pGAPZ, pA0815, and prokaryotes, mammals, yeast A gene component required for expression in a combination of gene components by a fungal or insect cell expression system or a heterologous expression system, or a combination of gene components from different expression systems so as to be expressed in at least one of the expression systems Include other vectors incorporating Align.
Recombinant vector screening
Transcription analysis Prior to scaling up the expression of the target protein, the vector containing the target sequence can be evaluated for correct transcription to the target. Accurate insertion of cDNA into cloning vectors is evaluated using in vitro, prokaryotic, eukaryotic or plant transcriptional expression systems in array form, and size analysis of purified transcripts It can be carried out. One preferred embodiment is shown in FIG. In this case, in vitro transcription analysis pre-screens vector constructs that may be used for expression and purification. Pre-selected vector constructs from cloning of inserts into suitable vectors can be placed in an array format shown in step S100, in this example a 3 × 6 array, and analyzed simultaneously. This vector construct can be selected from a variety of expression systems, examples of which include insect expression systems, plant expression systems (Geneware Vector®) (GWV) and bacterial expression systems (E. coli). It is done. If the insert DNA cannot be successfully inserted into one cloning, it may be useful to screen multiple clones from the cloned samples shown as A1-A6. The vector construct can include a T7 promoter located upstream of the cDNA insert or other promoter capable of in vitro transcription. T7 in vitro transcription shown as step S105 is started by adding bacterial T7TNA polymerase, and in step S110, the transcript length is analyzed by RNA agarose, polyacrylamide or other kind of RNA size separation gel electrophoresis or RNA analysis system. be able to. Record successful reaction S115 and unsuccessful reaction S120 according to the expected size of the transcript, and each (or the entire plate) if the number of transcripts within an acceptable range is below a predetermined threshold such as 50-75% ) The transcription reaction can be repeated. If the number of transcripts within an acceptable range is below the predetermined threshold described above, the clone is retransformed into a suitable host vector, followed by amplification and repurification of the T7 vector, followed by T7 RNA polymerase. Can be transferred.

Other transcription systems can also be used to evaluate successful transcription products within each cloned cDNA vector. Examples of such systems include SP6 transcription, T3 RNA polymerase, or any other transcription system. In any system, suitable promoters (SP6 and T3 promoters for each system) are required for recognition by RNA polymerase. After addition of RNA polymerase and transcription, the transcript can be analyzed by electrophoresis on polyacrylamide, agarose, or gel to determine whether it contains an appropriately sized transcript.
Once it has been confirmed that the target sequence for expression analysis has been correctly inserted into the vector, protein expression can be evaluated to determine the optimal vector and conditions for protein expression. Alternatively, the vector construct can be tested directly for protein expression, omitting all transcription and RNA analysis. In any form, an optimal protein expression system to be used in combination with the above-described protein purification method system can be determined using a small-scale protein expression evaluation as a screening method system.

  Assessment of protein expression can be performed in various expression systems such as plant, bacteria, yeast, fungus, insect and mammalian expression systems. A preferred expression system is to use a plant expression system for expression of the protein of interest. However, as mentioned above, some proteins do not express well in plant expression systems or do not express at all. Other expression systems can be convenient systems for expression depending on the type of equipment available for culturing and amplification of the host expression system. Other embodiments for testing vectors and inserts include bacterial, fungal, fermentative, insect and mammalian expression systems.

  Protein expression in plants can be assessed in various ways. One preferred embodiment according to the present invention is to evaluate protein expression both in the protoplast culture shown in step S130 and in the intact plant shown in step S125, as shown in FIG. An intact plant shown in step S125 can be infected with a suitable viral vector that expresses the target protein sequence (eg, GWV = Geneware Vector (registered trademark)). Preferably, young leaves and stems are infected with a viral vector containing the target sequence and surrounded by a protein shell. As shown in step S135, the viral vector is electroporated, bombarded (for example, a fine titanium or gold granule coated with a recombinant viral vector is driven into the cell at high speed), or the target protein sequence is Other methods of introducing heterologous nucleic acid to be expressed into intact plant cells can be inserted into the plant host. What is necessary is just to insert a plant vector in the small organic body of 1-3 pieces of 17-28 days old (preferably 21 days old). As shown in S2 of FIG. 1, a plant infected with a virus is grown for a predetermined time such as 10 to 16 days (but more preferably 12 days). Collect the grown plants and grind the infected leaves and stems with a twin roller drum to extract the homogenate at the same time, or any other method to finely grind the plant material Process by. The extract thus obtained is green juice, which is further processed to purify the expressed protein. For example, this green juice is mixed with 25 mM Tris pH 8.0, 500 mM NaCl, 2 mM PMSF, and 7 mM 8-mercaptoethanol buffer adjusted to 4% (w / v) PEG at a ratio of 1: 2. When the combination is allowed to stand at 4 ° C. for 1 hour and a half and then centrifuged, a green juice from which impurities are removed can be obtained. This green juice is added to a 96 well MBPP (melt blown polypropylene) filter plate containing 20 μl of Ni-NTA bead slurry. After incubating the green juice and Ni-NTA beads for 1 hour at room temperature, the green juice is removed from the well by spinning at 1000 × G for 5 minutes. The Ni-NTA beads are washed to remove non-specifically bound green juice proteins. The affinity labeled protein of interest is eluted from the bead by imidazole or EDTA incubation. The eluted protein is spun in a second 96 well plate and examined for the presence of the protein by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). As will be appreciated by those skilled in the art, green juice protein purification can be performed in a variety of steps, for example, in US Pat. No. 6,037,456 to Garger et al. The QIA expressionist® A Handbook for High-Level Expression of Purification and 6 × His-tagged process published in March 2001 by Qiagen, Valencia, California. This initial test can produce a small amount of expressed protein, which can be measured in μg or smaller units.

  For expression of the protein shown in S130 (FIG. 14), it may be desirable to transfect plant cell cultures or protoplasts with plant vectors rather than infect intact plants. Protoplasts are prepared according to standard molecular biology protocols for plant cell culture assays. Protoplasts can be derived from a wide range of sources, examples of which include leaves, buds, shoots, apex, or other available plant tissues. A preferred embodiment is a digest of leaf material obtained from Nicotiana tabacum. Other plants can also be used if they are suitable, depending on the type of vector selected for nucleic acid growth and protein expression. Examples include potatoes, Arabidopsis, other angiosperms or vascular plants, as well as various plants including moss and genus. Suspension with single cell protoplasts first disinfects the tissue surface using standard techniques such as collecting explant tissues such as leaves and exposing to sodium hypochlorite. The explant is then digested with an appropriate cocktail of enzymes to obtain a single cell suspension. In preferred embodiments, pectinases (Macerozyme R10, Pectolase Y23, Rhozyme HP150 and other pectinases) and cellulases (Cellulase, Cellulsin, Drisease and other cellulases) can be used. However, other enzyme cocktails that digest stromal tissue surrounding each plant cell can also be used.

  After enzymatic digestion, the protoplast suspension is placed in a microtiter plate (in this example a 2 × 3 microtiter plate array, but other microtiter plates can be used) and washed for a suitable medium. Incubate at Preferably, this is done with two sheets. In preferred embodiments, commercially available basic Murashige and Skoog media can be used, but the macro and micro components, soluble carbon sources, nitrogen vitamins and other growth factors necessary for the maintenance of protoplasts in vitro Other media preparations that provide an equilibrium mixture of can also be used. It is well known to those skilled in the art that many different combinations and ranges of media components can be successfully used for protoplast expansion.

  After suspension of the protoplast and incubation in a suitable medium, the protoplast cells can be transfected with DNA or RNA in a variety of ways. Preferably, the gene of interest is incorporated into a GENEWARE® vector, surrounded by a package or protein shell, and the surrounded virus is infected with the protoplast. Thus, the protoplast is transiently transfected with a suitable vector and induced in vitro to express the desired protein. Direct DNA microinjection, electroporation, liposome carriers, fine particle bombards (biolistics), silicon carbide fibers, or other methods can be used for the introduction and expression of foreign genes into plant cells.

  Alternatively, Agrobacterium tumefaciens-mediated Ti transfer can be used to introduce and express foreign DNA into the plant cell of cloned DNA. For example, the cloned DNA can be inserted into a suitable vector taken up by Agrobacterium. After insertion, protoplasts or intact plants are incubated in the presence of Agrobacterium containing DNA. Agrobacterium mediates transformation and integration into the plant cell host of insert DNA through the presence of the Ti gene. Subsequently, protein expression can be induced under the control of an inducible promoter cotransfected with the DNA of interest. Alternatively, a natural promoter that continues to regulate expression under a plant host can be transfected. Such natural promoters can include constitutively active promoters that can be modified to express foreign genes at high levels.

  Protoplasts can also be used as an initial screening tool to determine whether intracellular or secretory pathways are being used for protein expression. The microwell culture plate is first centrifuged to separate protoplast cells from the cell culture medium. The medium is aspirated and collected for parallel purification with protoplast cell lysate. Protoplast cell lysates and media, as well as homogenate suspensions from intact plants, are added to separate wells of a 96 well filter plate containing a metal binding matrix such as Ni-NTA beads or Ni chelating discs (Swell-Gel from Pierce) To do. The fluid between fractions is discarded and the metal binding matrix (such as Ni agarose) is washed with 40 mM imidazole / 0.5 M NaCl / phosphate buffer (pH 7.9). Next, the bound protein is eluted with 1M imidazole / 0.5M NaCl / phosphate buffer (pH 7.9) and analyzed on a 1D or 2D polyacrylamide gel (step S140 in FIG. 14). If an appropriately sized target protein has been produced, the target band is analyzed to quantify its expression level. Target proteins for protoplast samples are also noted for secreted or intracellular protein pathways, depending on which sample isolate contains the His-tagged protein.

  The target band can be cut from a 1D polyacrylamide gel as well as by identification with a 1D gel and analyzed with trypsin MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer). This device ensures accurate insertion of cDNA into a vector (accurate reading frame) and can confirm accurate protein expression. Trypsin MALDI can be performed by first eluting the protein from a polyacrylamide gel, digesting the protein with trypsin, purifying the fragment, lyophilizing and solubilizing in a suitable solvent. Thereafter, the sample is analyzed using a MALDI-TOF mass spectrometer or other ion desorption method, and peptide cleavage and mass measurement are sequentially performed. In another embodiment, the target band can be excised from a 1D polyacrylamide gel, or transferred to and eluted from a nitrocellulose or PVDF membrane. The isolated protein can then be sequenced using standard protein sequencing techniques (eg, Edman degradation, or other protein sequencing methods). In addition, after trypsin digestion of the isolated protein, standard protein sequencing techniques can be applied (eg, Edman degradation or other protein sequencing methods).

  After analysis on 1D polyacrylamide gel and MALDI-TOF MS, the probability that the expressed protein is the correct protein can be calculated using standard database analysis.

  As described above with reference to FIG. 13, as shown in FIG. 14, the analysis in step S <b> 140 is used to determine whether or not the expressed protein is acceptable in step S <b> 145 and to determine the failure in step S <b> 150.

  Vectors and their inserts can also be evaluated using bacterial, fungal, yeast, insect and mammalian expression systems. For example, if no protein was expressed from a transfected protoplast culture or an infected plant, the corresponding MEV cDNA was analyzed for expression in E. coli and the cause of the DNA transfection error in the plant or protoplast was not expressed. It can be confirmed that it is not. Alternatively, bacterial expression systems, fungal expression systems, yeast expression systems, insect expression systems may be tested in parallel with plant expression systems to determine the optimal expression system for protein expression and purification.

  There are a number of methods well known to those skilled in the art for expressing foreign proteins from a cDNA vector in a prokaryotic host. Preferred embodiments include transformation in a 96 well format of a suitable host strain of E. coli, such as NovaBlue DE3, using a MEV vector containing a cDNA or genomic DNA insert. This transformant can be placed on a solid medium containing an antibody selected according to the vector used and grown overnight at 37 ° C. in a 96 deep well block. The grown E. coli culture is diluted in fresh medium containing isothiopropyl galactoside (IPTG) and protein expression is induced through the □ -galactosidase promoter in the vector. Alternatively, other strains of E. coli or suitable prokaryotic host strains may be propagated with other vectors containing vector DNA and its inserts, and alternative inducible promoter systems such as temperature dependent expression or other inducible expression systems. Can be used for

After logarithmic growth for a defined time, 2 microliters of culture can be spotted onto an 8 × 12 grid of nitrocellulose membrane and Western blots can be performed using antibodies against the target protein or tag. Alternatively, the expressed protein and the hexa-histidine tag attached thereto can be isolated and purified on the Swell-Gel Ni chelate disk matrix of the 96 well filter plate described above. The eluted protein can be analyzed on a 1D polyacrylamide gel to determine if the expression was of an appropriate size. Alternatively, trypsin MALDI-TOF can be performed on the excised protein band for further identification.
Protein expression scale-up After protein evaluation and screening, protein expression and purification can be scaled up. As shown in S3 of FIG. 1, the protein evaluation includes confirmation that the desired protein was expressed, the amount of plant obtained per plant, and the expression level of the target protein. Next, as shown in S4 of FIG. 1, using the obtained plant quantity and target protein expression level, the number of organisms (ie, tobacco plants) necessary for production of a desired quantity of target protein is calculated. As shown in S4 of FIG. 1, the required number of organisms (ie tobacco plants) for the production of the desired amount of target protein are planted and infected with the transgenic virus and protein that are to be expressed therein. Let It should be understood that a series of steps similar to step S1 to step 4 can be applied to the use of mammalian, yeast, insect or bacterial protein production systems.

  Steps indicated as S6 and S7 in FIG. 1 will be described in detail with reference to FIGS. As shown in S10 of FIG. 3, a plurality of types of proteins can be expressed in a selected expression system such as a GENEWAREX (registered trademark) expression system.

  A tobacco plant is collected and decomposed inside a Waring blender, a commercially available juicer, etc., and as shown as S11 in FIG. 3, one or more desired proteins are released as green juice from the leaf cells. Let Usually the ratio of biomass to extraction buffer is 1: 2, which can be vacuum infiltrated into the plant material before extraction. A typical extraction composition is 25 mM Tris pH 8.0, 500 mM NaCl, 2 mM PMSF, and 7 mM 8-mercaptoethanol to which 1% (w / v) or less Tween-20 and 5% ( w / v) The following sodium ascorbate may be contained. Next, as shown as S12 in FIG. 3, the green juice is treated with a cleaning agent such as polyethylene glycol (PEG) (concentration is 300 mM to 2 M), which is usually contained at 4% (w / v) in NaCl. . However, it should be understood that detergents such as polyvinylpyrrolidone (PVPP) may be used alone or in combination with PEG. PEG is a smaller size protein that allows the inventors to remove more significant chlorophyll-containing proteins and membrane complexes, making the green juice sufficiently transparent to allow loading into a chromatography column. (And the target protein) has been found to be a detergent that suspends in green juice. Specifically, when PEG is added to green juice, which is an aqueous solution, more proteins interact, and the aggregates are easily discharged from the solution by centrifugation or filtration.

  The green juice treated with the detergent can be treated in at least one of two ways. The first method is to subject the green juice treated with PEG to filtration, as shown in S13 of FIG. In this treatment, a greenery juice is mixed with a filter aid such as pearlite (volcanic rock), and the final concentration is 1% (w / v) to 10% (w / v), preferably 4% (w / v). v). The green juice is then passed through a glass fiber filter having an average pore size of 1.2 microns and coated with pearlite. Green juice is clarified and passes, but perlite and larger protein aggregates remain in the filter. Next, the step shown as S15 of FIG. 3 can be given to the purified green juice. However, it should be understood that the step shown as S15 in FIG. 3 is optional and not essential.

  Another method was to purify larger protein aggregates as shown as S14 in FIG. 3 by centrifuging the green juice treated with PEG for approximately 20 minutes at a force of 3700G after step S12. Separate from green juice. Filtration through the Miracloth continues to remove debris that does not become fine. Thus, it can be seen that in addition to producing green juice suitable for chromatography, both purification methods similarly reduce the infectious virus titer.

  If necessary, the purified green juice is subjected to a freezing and thawing process shown as S15 in FIG. Specifically, the purified green juice is frozen and thawed in step S13 or step S14, and then re-centrifugated as shown in step S16 of FIG. By freezing and thawing treatment, the starchy material and other contaminating plant proteins are precipitated, and further subjected to centrifugal treatment or filtration treatment to separate them from the purified green juice. This step S15 is arbitrarily performed according to the transparency of the green juice after filtration or centrifugation, and assists the downstream purification step, but is not an essential step in the present invention.

Next, the amount of purified green juice is normalized so that multiple types of samples containing different proteins can be purified simultaneously. For normalization, urea or glycerol can be added to bring the sample to a predetermined concentration and / or pH adjustment value. For example, the concentration of urea is 50 mM to 4 M, the concentration of glycerol is 5% (w / v) to 50% (w / v), and NaOH (sodium hydroxide), sodium phosphate, or Tris buffer is used. The pH can be increased from 7.2 to 7.3 to 7.5 to 8.0. It should be understood that only pH adjustments can be made during normalization. Various amounts of urea and glycol may or may not be included depending on the desired target protein characteristics and performance.
As shown in step S17 of FIG. 4, the purified and normalized green juice is put into a purifying apparatus such as the apparatus described below with reference to FIGS.
Here, the description of the method of the present invention shown in FIG. 4 is matched with the description of the apparatus shown in FIGS.

  As shown in FIG. 5, the purifying apparatus of the present invention is a feeding reservoir 5 in which the purifying apparatus of the present invention is first filled with purified green juice and buffer solution as shown in step S <b> 17 of the process flow diagram of FIG. 4. including. During the purification process, the feed reservoir 5 is immersed in a larger container 10 filled with a coolant such as a mixture of water and ice. This maintains the feed reservoir 5 and the purified green juice at a temperature below 10 ° C., preferably about 4 ° C., more preferably near 0 ° C., but above the freezing point of the green juice. In order to minimize oxidation and protease activity, it is desirable to maintain the clarified green juice at approximately low temperatures. It should be understood that any coolant, such as water and ice, can be used in container 10 to maintain the temperature of the clarified green juice above the freezing point, but below 10 ° C. Alternatively, it is possible to maintain the periphery of the container 5 at a low temperature by using a refrigeration mechanism. Although not shown, feed reservoir 5, larger container 10, and flow through collection reservoir 70 (described below) are placed in a robotic fluid handler to operate fluids in a more automated manner. May be. Any of a variety of robotic fluid handling devices can be used as such fluid handlers, for example, the robotic sample processor Genesis RSP, the modular automatic workstation Genesis Freedom, or the automatic workstation Genesis Workstation. Devices manufactured and sold by TECAN of Zurich, Switzerland.

  The feed reservoir 5 is connected to a tube 15, which is connected to a first valve 20. The first valve 20 is connected to a tube 25, and this tube 15 is further connected to a pump 30. The pump 30 may be any of various pumps, but is preferably a low-speed pump that allows the purified green juice to pass through the purification apparatus of the present invention at a substantially low speed. For example, the pump 30 can be an ISMATEC (registered trademark) variable speed pump peristaltic pump having a flow rate range of 0.01 to 44.4 mL / min. This pump is also a multi-channel pump that can simultaneously purify plural kinds of proteins by the method described below with reference to FIG.

  This pump 30 is connected to the second valve 40. The second valve is connected to a tube 45, which is connected to the column 50. The column 50 is connected to a pipe 55 connected to the third valve 60. The third valve 60 is connected to a tube 65 that is connected to a flow through collection reservoir 70. From the following description, the green juice filled in the supply reservoir 5 passes through the tubes 15, 25, 35, 45, 55 and 65 from the supply reservoir 5 through the injection operation of the pump 30, and the column It should be understood that 50 and valves 20, 40 and 60 are received in collection reservoir 70.

  The column 50 has a porous frit that allows a fluid stream to penetrate while retaining the material therein, thereby bringing the fluid flowing into contact with the retained material and causing an interaction therebetween. It is equipped. In the purification apparatus of the present invention, this material in the column 50 is used to temporarily hold a desired target protein, for example, an affinity resin such as the example marketed as Qiagen ™, or the like. Material. When the purified green juice is flowed into the column 50, the target protein adheres to the affinity resin and is retained.

  Valves 20, 40 and 60 are connected to tubes 75, 80 and 85, respectively, and are included in the purifier for various purposes. In the purification mode, the valve 20 is set so as to allow fluid to communicate (fluid flow) from the tube 15 to the tube 25. Valve 20 is set to allow fluid to flow from tube 75 to tube 25, such as to remove washed and purified target protein (this will be described below) or to prepare for equilibration of pump 30 and system. It is also possible to do. The valve 20 can also be set to allow fluid to flow from tube 15 to tube 75.

In the purification mode, the valve 40 is set to allow fluid to communicate from the tube 35 to the tube 45. However, it is also possible to set the valve 40 so that the fluid flows from the tube 35 to the tube 80 for the purpose of cleaning or preparing the pump 30. Alternatively, the valve 40 may be set so that the fluid flows from the tube 80 to the tube 45 for the purpose of washing the column 50 or removing the purified target protein.
In the purification mode, valve 60 is typically set to allow fluid to communicate from tube 55 to tube 65. It is also possible to set the valve 60 so that the fluid flows from the tube 55 to the tube 85 for the purpose of washing the column 50 or removing the target protein purified in the column 50. Valve 60 may be set to allow fluid to flow from tube 85 to tube 65 to allow tube 65 to be flushed and washed.

  It should be understood that the valve 40 is an optional component and may alternatively be omitted from the apparatus shown in FIG. 5 depending on the application of the apparatus.

  Depending on the environment in which the system is deployed, it may be necessary to prepare the system for startup. Specifically, by operating valves 20 and 85, fluid can be introduced from vessel 100 into line 15, line 25, pump 30, line 35, line 45 and line 55. Typically, this system is prepared by directly interconnecting lines 45 and 55 with column 50 removed. When this preparation is completed, the removable column 50 is inserted again between the line 45 and the line 55 as shown in FIG. In this preparation step, the tube 15 is filled with a preparation fluid. It should be understood that there is no green juice in the reservoir 5 at the stage of preparation, and that the green juice can be injected into the reservoir 5 via the automatic fluid handler when preparation is complete. Lines 45 and 55 may have special connections (not shown). Thereby, the column 50 can be easily attached and detached during the preparation process.

  In the operation of this purification system, the purified green juice is put into the juice container 5. After the charging, the pump 30 is operated to draw the purified green juice from the juice container 5 and pass through the tube 15 and the valve 20, and of course, the pump 30, the tube 35, the tube 45 and the valve 40, and into the column 50. Let it flow. In the column 50, the purified green juice interacts with the material disposed in the column 50, ideally all the target protein is retained in the column 50, and the remaining green juice portion is essentially from the column 50. Spills as waste. This waste liquid juice passes through the tube 55, the tube 65 and the valve 85 and flows into the collection reservoir 70.

  Returning to FIG. 4, the conditions of the affinity resin and the column 50 before the start of the purification mode must be adjusted before purification, as shown as “column equilibration” in S <b> 18 of FIG. 4. In order to equilibrate the column 50, as shown in FIG. 8, an equilibration solution that stimulates the properties of the green juice and the buffer in the container 5 is introduced into the container 100. For example, the pH of the equilibration solution is usually equal to the clarified green juice and buffer, and urea, PEG and / or glycerol are included in the equilibration solution at the same concentration if contained in the green juice and buffer. . As shown in FIG. 8, this equilibrated solution is passed through the column 50 from the container 100 and discarded through the pipe 85.

Thereafter, the valves 20 and 60 are set to the purification mode, and the purified green juice and buffer mixture are injected from the container 5 through the column 50 into the collection reservoir 70 as shown in S19 of FIGS. As described above, the affinity resin captures the target protein by interaction with the tag in the protein. The purified green juice and buffer mixture are passed through the column 50 at a predetermined speed by the pump 30 so that the residence time in the column 5, that is, the affinity resin is 30 seconds to 5 minutes. However, preferably, the green juice and the buffer mixture are injected by the pump 30 at a constant speed at which the residence time of the green juice in the column is approximately 1 minute.
After the entire mixture has passed through the column 50, contaminants must be washed away and certain buffer components, such as PEG and urea, removed as shown in step S20 of FIG. As shown in FIG. 10, the contaminant is washed out from the column 50 through at least one of the two types of solutions stored in the container 105 and the container 110 via the proportioning valve 115. For example, the interaction between the contaminated protein and the affinity resin can be reduced by using a buffer in the container 110 containing a low concentration of the competitive inhibitor imidazole (10 to 90 mM) for various proteins. Alternatively, the solution in container 110 is initially made to contain urea, glycerol and / or PEG at a concentration similar to the clarified green juice. This is passed through the column 50 and its flow rate is gradually decreased linearly as the flow rate from the container 105 is increased linearly. The buffer in reservoir 105 contains different concentrations, urea, glycerol and / or PEG, which concentration is usually zero. Therefore, the concentration of unnecessary components gradually decreases during this process. If the state in the column 50 changes abruptly, the retained labeled protein may be adversely affected, but this prevents the state in the column 50 from changing rapidly. As shown in FIG. 10, the washed out component is discharged through the pipe 85.

Next, as shown in step S21 of FIG. 4, the column 50 is eluted, the target protein is taken out, and fed into the reservoir as shown in FIG. Usually, a predetermined eluate such as phosphate buffered saline containing 100 to 200 mM imidazole or EDTA is supplied into the reservoir 127 shown in FIGS. As shown in FIG. 11, the solution in the reservoir 127 is pumped from the column 50 via valves 90 and 20 to release the captured target protein from the affinity material in the column 50, which is stored in the reservoir 118. Capture in.
Alternatively, before elution from the column 50, after washing, reconstitution buffer (eg, phosphate buffered saline, Tris buffered saline, or other buffer used for reconstitution) is introduced in a linear gradient. Thus, the target protein may be refolded in situ on the column matrix. For example, when a number of histidine-tagged proteins are purified under denaturing conditions, the histidine tag is exposed at the carboxy terminus or amino terminus, so that the amount of binding of this tag to the linking group contained on the metal affinity matrix is reduced. To increase. The histidine-tagged protein is subsequently eluted from the metal affinity matrix in a denatured state by lowering the pH of the buffer that passes through the column or by introducing high concentrations of imidazole or EDTA. The eluted protein may fall or settle out of solution, especially at higher concentrations. This is thought to be due to intermolecular interactions between exposed hydrophobic groups due to the denatured state of the eluted protein. If the protein cannot be redissolved, the overall yield of protein is reduced. However, by introducing a recovery buffer in a linear gradient after washing, the protein may be able to refold while bound to the affinity matrix. During refolding, previously exposed hydrophobic groups are masked, thus avoiding hydrophobic interactions between molecules and protein precipitation.

  It is important to use a gradient to induce protein refolding. The method claimed by the present application does not change its implementation according to the understanding of the mechanism by the present application. However, by gradually introducing a restoration buffer, the protein is properly folded while binding the protein to the affinity matrix. In assistance, the bound protein may be given time to properly refold into a complex tertiary or quaternary structure. After refolding the target protein on the column, an elution buffer can be introduced to elute the protein from the column.

  A linear gradient maker can be used to bind to the affinity matrix while refolding the target protein in situ. Using a linear gradient maker, the reconstitution buffer can be gradually introduced over a certain amount or time. A linear gradient maker with at least one pump or proportioning valve to draw its fluid from two reservoirs, including the starting buffer and the final buffer, such as reservoirs 105 and 110 shown in FIGS. Can do. For example, a denaturation buffer can be included in the first reservoir and a reconstitution buffer can be included in the second reservoir. An adjustment valve or a proportioning valve 115 located between the first reservoir and the second reservoir adjusts the inflow amounts of the two types of buffer solutions and flows from the second reservoir to the first reservoir. Change the composition of the column running buffer. The composition of the column running buffer mainly consists of a denaturing buffer at the beginning of running. This buffer composition gradually changes with the introduction of the restoration buffer from the second reservoir to the first reservoir, and finally the column running buffer contains only the restoration buffer and the protein is gradually reduced. Fold. Alternatively, a mixing chamber (not shown) can be used. In this case, the solutions in the first and second reservoirs are injected into the mixing chamber and then pass through the column. Similar to the above, the relative ratio between the first reservoir and the second reservoir changes, and the composition of the running buffer is mainly composed of denaturing buffer at the start of running and mainly from the restoring buffer at the end of running. Become. Upon protein folding, the labeled protein can be removed from the affinity matrix by passing through an elution buffer.

  The restoration buffer can be gradually introduced step by step instead of a linear gradient. For example, a buffer with a reduced salt or urea concentration can be flowed stepwise into the column. It will be apparent to those skilled in the art that there are various ways to gradually introduce reconstitution buffer and perform the refolding of the denatured target protein in situ in the affinity matrix.

  There are several types of proteins that are difficult to separate from each other. Therefore, in the alternative embodiment shown in FIG. 6, a sacrificial column 46 may alternatively be added to the apparatus shown in FIG. Specifically, in FIG. 6, the sacrificial column 46 is connected to the tube 45 so as to be in fluid communication with the tube 45 so that all the liquid juice flowing out from the tube 45 flows into the column 46. The column 46 is further connected to a tube 47 for fluid communication therebetween. The tube 47 is connected to the valve 48, the valve 48 is connected to the tube 49, and the tube 49 is connected to the column 50 described above. All the other components of the system shown in FIG. 6 are the same as those of the embodiment shown in FIG.

  Valve 48 is also connected to tube 90. In the purification mode, the valve 48 directs the juice from the tube 47 to the tube 49 and causes it to flow into the column 50. However, it is also possible to set the valve 48 so that the tube 47 and the tube 90 are in fluid communication. In addition, the valve 48 can be set in fluid communication between the tube 90 and the tube 49 for the purpose of removing washed, flushed or purified protein as described below.

  In addition, as shown in FIG. 9, a circulation valve 200 may be provided in this apparatus so that the green juice can pass through the column 50 a plurality of times.

As shown in FIG. 12, a computer is provided to automatically control each embodiment of the apparatus according to the present invention shown in FIGS. Specifically, the computer is connected to the device pump and various valves. It should be understood that the above description regarding the operation of the system shown in FIGS. 5 and 8-11 is also applicable to the apparatus of FIG. 6 and the apparatus of FIG.
The apparatus of FIG. 7 shows a system having a dedicated column 50 with a plurality of separate flow channels. Each column 50 operates in parallel to simultaneously purify a plurality of types of proteins. Specifically, since the single peristaltic pump motor M connected to each of the pumps 30 can inject a plurality of flow channels, the green juice can be simultaneously flowed into the plurality of columns. Each of the feed reservoirs 5 is immersed in a single ice bath. The desired column residence time is obtained by operating the peristaltic pump motor. Thereby, as above-mentioned, target liquid can be capture | acquired from green juice with high reliability because green juice flows in the column 50 at a fixed speed. Due to the slow flow rate, it can take a considerable amount of time to obtain an acceptable purification of the target protein. If there is only one flow channel such as the flow channel of the apparatus shown in FIG. 5, it takes an enormous amount of time to purify multiple types of proteins. Therefore, by providing a plurality of flow channels in a single apparatus in parallel, a plurality of types of proteins can be extracted simultaneously, which is a great advantage for the protein purification step.

  The computer shown in FIG. 12 is connected to the motor M of the pump 30, or alternatively to the single pump 30, valves 20, 40, 60, 90 and 115. This computer can be further connected to a temperature sensor T and a pressure sensor (not shown) to control the multi-chamber system. Alternatively, pressure sensors can be provided on the apparatus downstream and upstream of the column 50 to control fluid flow between them. In addition, in the alternative embodiment shown in FIGS. 6 and 9, a computer may be connected to valves 44, 48 and 200 to provide these controls.

  The computer shown in FIG. 12 is a part of the LIMS (laboratory information management system) shown in the block diagram of FIG. 1, and is connected to the server shown in FIG. 2 by a LAN (local area network). As shown in FIG. 1, LIMS is an integrated part of the process according to the present invention, gene sequence, DNA sequence used for protein expression, expressed protein, amount of each protein produced, expression used for production of such protein. Includes software to track all biological materials such as systems, all data related to the prescreening process, correlation of searched database information, and various steps performed to produce and purify the protein of interest It is out. The LIMS includes the computer system shown in FIG. 2 and the computer shown in FIG. 12, which further includes various valves 20, 40, 60, 90 and 115 and optional valves 44, 48. And 200 to control to allow the protein of interest to be isolated and eluted as described above.

According to the present invention, it is possible to purify a large amount of protein in milligram units by a method having both good cost performance and efficiency. The method and apparatus according to the present invention fulfills one need because other methods and apparatus can be used to purify very large amounts (100 g to Kg units) or very small amounts (μg units).
Example 1: Geneware (registered trademark) system in which a subset of proteins with an emphasis on labeling in which expression and purification of multiple types of proteins for antibody production are restricted to the lung or brain are performed in parallel Was selected for expression and subsequent purification. Protein databases such as the Human Protein Index (HPI) and Swiss Spot were examined for potential proteins and subsets selected based on the availability of full-length clones from in-house and commercial gene collections. One sequence ID was assigned to each full-length clone so that the DNA sequence and the resulting protein could be tracked in the laboratory information management system (LIMS) from vector generation to confirmation (FIG. 1). Using the polymerase chain reaction (PCR) containing primers complementary to the DNA sequence, the reading frame of each target was amplified and purified and ligated into an appropriate GENEWARE® expression vector. This vector was modified to include a histidine tag sequence, so that the expressed protein had a tag at the N-terminus. The resulting vector was screened to confirm insertion integrity and coordination. Successful cloning events were evaluated for protein expression, and failed events were reintroduced into the cloning workflow.

  For screening, sufficient in-vitro transcription was generated for each clone to inoculate three 21-day-old tobacco plants. 12 to 14 days after inoculation, plant material located above the inoculated leaves was collected, weighed, and dissociated to obtain green juice. Combine 1 volume of green juice with 2 volumes of extraction buffer (25 mM Tris pH 8.0, 500 mM NaCl, 2 nM PMSF, 7 mM 6-mercaptoethanol) in a deep well block (96 well) 4% (W / v) PEG (final volume 1500 μl) was adjusted to stimulate the extract obtained during protein production. After being stored at 4 ° C. for 1 hour and a half, the green juice was centrifuged at 3000 × G for 20 minutes to obtain a purified green juice containing the target protein. To capture the target protein, 700 μl of clarified green juice was combined with 25 μl of affinity resin (QiagenNi-NTA) in a 96 well filter plate and incubated for 1 hour at room temperature. The green juice was removed after incubation using a sufficiently hydrophobic filter to hold the purified green juice, and centrifuged at 1000 × G for 5 minutes. The affinity resin combined with the captured protein was retained on the filter and washed twice with 700 μl wash buffer (16 mM Tris pH 8.0, 330 mM NaCl, 5 mM imidazole). Centrifugation was performed at 1000 × G for 5 minutes between this washing. Recovery of the target protein from the affinity resin is accomplished by incubating the resin for 5 minutes in 60 μl elution buffer (16 mM Tris pH 8.0, 150 mM NaCl, and 200 mM imidazole or 200 nM EDTA) and centrifuging (1000 XG for 5 minutes) and recovering the eluate. This elution step was repeated to obtain 120 μl of final product. In order to evaluate the expression level of each labeled protein, the eluate obtained from each purification was analyzed by SDS-PAGE. After Coomassie staining, a protein band almost identical to the correct molecular weight was seen (± 20%), and if no co-migrating band was seen in the negative control, the target protein was expected to be well expressed. The protein level was quantified by concentration measurement using a bovine serum albumin standard. This variable was entered into the LIMS system along with the recorded plant quantity to determine the number of plants required for target protein production.

  Nine sets of wild tobacco plants were planted for protein production. To facilitate tracking and inoculation, the number of plants required for each target protein was rounded up to the nearest multiple of 9. The expression level of each protein varies significantly, resulting in a significant change in the number of plants required to achieve a given protein level. Therefore, 9 to 96 21 day old plants were typically used and the in-vitro transcription reaction scaled up accordingly. After 12-14 days after inoculation, plant material located above the inoculated leaves was collected, weighed and combined with 2 volumes of cooled extraction buffer. This extraction buffer was vacuum infiltrated into the plant material so that the buffer / plant material was evenly distributed. Thereafter, green juice was obtained using a commercially available juice extractor. PEG was added to 4% (w / v) and the green juice was left at 4 ° C. for 1 hour and a half to flocculate and settle the chlorophyll-containing component of the extract. The green juice was purified by filtering using 4% (w / v) perlite as a filter aid. The clarified green juice was adjusted to 10% (w / v) glycerol to minimize the interaction of the hydrophobic protein with the affinity resin and the extraction volume was normalized with the extraction buffer. Each channel of the pre-equilibration purifier was loaded with clarified green juice containing a specific target protein. When the amount of green juice for a given target protein was substantially higher than for other target proteins, the clarified green juice was divided into two or more channels. The purified protein was eluted from the affinity resin and stored. The purified green juice was passed through the affinity resin, and the histidine-labeled protein was retained on the Ni-NTA affinity resin. 10 column volumes of wash buffer were passed through the column to remove contaminating plant proteins and the target protein was recovered with an elution buffer containing 200 nM EDTA. The composition of this extraction buffer, wash buffer, and elution buffer was the same as that used in the screening step. An aliquot of each eluate was analyzed by SDS-PAGE and densitometry of the protein band subjected to Coomassie staining to determine the protein concentration. If necessary, the protein was concentrated by ultrafiltration and the total protein was dialyzed into phosphate buffered saline. Thereafter, this was stored at 20 ° C. SDS-PAGE was performed on the final protein that had been concentrated and dialyzed to determine protein purity, and trypsin MALDI was performed to confirm protein identification.

  Tables 1A and 1B summarize the results of the production process, in which 5-15 unique proteins were expressed using GENEWARE® and purified in parallel. Based on the screening, a sufficient amount of plants was inoculated to obtain a minimum of 9 plants per target protein and 1.5 mg of purified protein. In production mode, 10 out of 27 targets achieved more than the required protein level. In the case of 11 types, when the number of plants was appropriately adjusted and the second production was performed so as to satisfy the protein requirement, no protein was recovered with 6 types of targets. This result was confirmed by performing the GENEWARE expression system on 9 sets of plants. If the protein is not recovered after this purification, its sequence ID is distinguished as GENEWARE incompatible and evaluated with another expression system such as a mammalian expression system.

本発明の種々の詳細について、本発明の趣旨および範囲を逸脱することなく、さまざまな変更を加えることが可能である。また、本発明による実施形態に関する上記説明は、例示のみを目的としており、請求の範囲およびその等価物により規定される本発明を何ら制限するものではない。

Various modifications can be made to the various details of the invention without departing from the spirit and scope of the invention. Further, the above description of the embodiments according to the present invention is for illustrative purposes only and does not limit the present invention defined by the claims and their equivalents.

FIG. 2 is a process flow diagram illustrating the general steps of a highly flexible method for producing and purifying one or more types of predetermined proteins according to the present invention. FIG. 2 is a schematic diagram showing a portion of a computer system used in an embodiment of the present invention, which assists in the selection of proteins and the identification of genetic sequences that express one or more selected proteins. To do. 2 is a process flow diagram illustrating the steps of a method for purifying one or more types of proteins produced according to the present invention. FIG. FIG. 4 is another process flow diagram showing successive steps of the purification method shown in FIG. 3 according to the present invention. 1 is a schematic diagram showing components of an apparatus for purifying a single protein according to the present invention. FIG. 2 is a schematic diagram showing components of an alternative embodiment of an apparatus for purifying a single protein according to the present invention. FIG. 6 is a schematic diagram illustrating a plurality of devices, such as the device shown in FIG. 5, operating in parallel to simultaneously purify multiple types of proteins according to the present invention. FIG. 6 is a schematic diagram showing the operational steps of the apparatus shown in FIG. 5 with the equilibration solution being passed through the apparatus according to the invention. FIG. 9 is a schematic view similar to FIG. 8 showing another operational step with green juice being passed through the device to capture the protein of interest in the column of the device according to the present invention. FIG. 10 is a schematic view similar to FIGS. 8 and 9 showing another operational step with the wash liquid being passed through the apparatus to wash away unwanted material from the column according to the present invention. FIG. 11 is a schematic view similar to FIGS. 8 to 10 showing a state in which an eluate is passed through a column in order to take out a target protein from the apparatus according to the present invention. It is the schematic which shows the part which controls the refinement | purification apparatus by this invention among the computer systems used by one Embodiment of this invention. It is the schematic which shows the prescreen for correctly transcribe | transferring several types of vector using in vitro transcription | transfer and gel electrophoresis analysis. A vector that expresses the correct size transcript for gel electrophoresis is used in subsequent studies to determine the optimal vector and system for protein purification. FIG. 2 is a schematic diagram showing pre-screening to accurately translate and express multiple types of vectors using intact plants and / or cell culture protoplast systems.

Claims (20)

A method for purifying multiple types of proteins,
Expressing a first labeled protein using a first predetermined organism;
Expressing a second labeled protein using a second predetermined organism;
Collecting the first labeled protein from the first predetermined organism to form a first green juice;
Collecting the second labeled protein from the second predetermined organism to form a second green juice;
In order to extract the first labeled protein, the second green juice is extracted in order to extract the second labeled protein simultaneously with injecting the first green juice into the first affinity column. Passing through a second affinity column. Collecting the first green juice after passing through the first affinity column;
Collecting the second green juice after passing through the second affinity column;
Circulating the second collected green juice into the second affinity column simultaneously with circulating the first collected green juice into the first affinity column. The method according to 1. Recovering the first labeled protein from the first affinity column;
Recovering the second labeled protein from the second affinity column,
The recovered first labeled protein can be measured in an amount ranging from 1 μg to 100 mg, and the recovered second labeled protein can be measured in an amount ranging from 1 μg to 100 mg. The method of claim 1.   The method according to claim 1, wherein the predetermined organism comprises one or more of organisms composed of bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells.   The method according to claim 1, wherein each of the labeled proteins is a different type of recombinant protein used for constructing a plurality of microarrays.   The method of claim 1, wherein each of the labeled proteins is a different type of drug specific to a patient. A method for producing and purifying multiple types of proteins, comprising:
Selecting at least two protein targets;
Selecting a suitable recombinant vector that expresses the exact protein sequence of the protein target;
Expressing each of the selected recombinant vectors in a separate predetermined organism to produce a respective recombinant protein;
Collecting the organism to form at least two types of green juice corresponding to each of the recombinant proteins;
Injecting the plurality of types of green juice into each affinity column simultaneously to extract the recombinant protein;
Including methods. Collecting the plurality of types of green juice in separate containers after passing through the plurality of affinity columns;
The method of claim 7, further comprising circulating the plurality of types of green juice in the plurality of affinity columns simultaneously. Recovering the recombinant protein from the affinity column;
The method according to claim 7, wherein the recovered recombinant protein can be measured in an amount ranging from 1 μg to 100 mg.   8. The method according to claim 7, wherein each of the predetermined organisms includes one or more organisms composed of bacterial cells, yeast cells, insect cell cells, plant cells, and mammalian cells.   The method according to claim 7, wherein each of the recombinant proteins is a different type of recombinant protein used for manufacturing a plurality of microarrays.   8. The method of claim 7, wherein each of the recombinant proteins is a different type of drug specific to the patient. An apparatus for purifying multiple types of proteins,
Multiple reservoirs each containing different types of green juice,
A plurality of pumps, one connected to each of the reservoirs;
A plurality of columns connected to each one of the corresponding pumps and having protein-retaining material disposed therein;
Means for removing the protein from the column;
Including the device.   The apparatus of claim 13, further comprising a computer connected to portions of the apparatus for automatically controlling the apparatus. An apparatus for purifying multiple types of proteins,
A first reservoir containing a first green juice;
A first pump to which the first reservoir is connected;
A column connected to the first pump and having a protein holding material disposed therein, wherein the first green juice is pushed out by the pump and passes through the column;
A computer connected to the pump for automatically controlling the device. An apparatus for purifying multiple types of biological materials,
A first reservoir containing a first solution;
A second reservoir containing a second solution;
A first valve connected to the first reservoir;
A second valve connected to the second reservoir;
A first column having a biological material-retaining material disposed therein and connected in fluid communication with the first valve to direct the first solution into the first column; 1 column,
A second column having a biological material-retaining material disposed therein and connected in fluid communication with the second valve to direct the second solution into the first column; Two columns;
A computer connected to the first valve and the second valve for automatically controlling the apparatus, and a flow path passing through the first column and a flow path passing through the second column; Are isolated from each other and are capable of flowing the solution simultaneously into the first column and the second column. A third valve located downstream from the first column for controlling fluid flow exiting the first column;
A fourth valve located downstream from the second column for controlling fluid flow exiting the second column;
The plurality of types according to claim 16, wherein the third valve and the fourth valve are connected to the computer so that the computer can control the operations of the third valve and the fourth valve. For the purification of biological material. A first pump located upstream of the first valve;
A second pump located upstream of the second valve;
Further including
18. The biological material of claim 17, wherein the first and second pumps are connected to the computer so that the computer can control the operation of the first and second pumps. Equipment for purification.   The apparatus for purifying biological materials according to claim 18, wherein the first and second pumps are connected to a single motor, and the operation of the motor is controlled by the computer. The first reservoir, the first valve, the first column, the third valve located downstream of the first column, and the first pump all define a first flow path. And
The second reservoir, the second valve, the second column, the fourth valve located downstream of the second column, and the second pump are all in the first flow path. A second flow path provided separately from the
In order to purify another biological substance in parallel with the first flow path and the second flow path, the device is separate from the first flow path and the second flow path. The apparatus for purifying a plurality of types of biological substances according to claim 18, further comprising a third flow path provided.

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